Complete inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase
Complete inhibition of Na+, K+, Cl- cotransport in Madin-Darby canine kidney cells by PMA-sensitive protein kinase
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DOI：
10.1016/s0005-2736(97)00225-3
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发表时间：
1998-03-02
影响因子：
3.4
通讯作者：
Hamet, P
中科院分区：
文献类型：
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作者：
Gagnon, F;Orlov, SN;Hamet, P
本研究使用具有低跨上皮电阻（194 +/- 47 Ω/cm（2））的Madin-Darby犬肾细胞，检测了激素和神经介质在肾上皮细胞Na+、K+、Cl-共转运调节中的作用。在该细胞系中，P-2-嘌呤受体激动剂可抑制Na+、K+、Cl-共转运（测量为布美他尼敏感性Rb-86内流）高达50- 60（ATP近似ADP > UTP > AMY），通过cAMP信号传导激活剂略微增加（15-30%）（forskolin，8-Br-cAMP），并且对cGMP信号传导的激活剂（8-Br-cGMP，硝普钠）、EGF、血管紧张素II、缓激肽、乙酰甲胆碱、普萘洛尔、加压素、腺苷、多巴胺和组胺不敏感。用0.1 μ M PMA预孵育MDCK细胞30分钟，可完全阻断Na+、K+、Cl-共转运的活性，而用1 μ M PMA预孵育24小时，下调该酶的活性，则可使Na+、K+、Cl-共转运激活60%，并消除PMA短期处理的效应。ATP对Na+、K+、Cl-共转运的调节对PMA敏感的蛋白激酶C亚型的下调不敏感。此外，在蛋白激酶活性的抑制剂，星形孢菌素，取消了0.1 μ M PMA的效果，但不改变ATP抑制这种载体。因此，这些结果首次表明，P-2-嘌呤受体和PMA敏感的蛋白激酶C亚型在MDCK细胞的Na+，K+，Cl-共转运的调节中起着关键作用。这些结果还表明，无论是PMA-或星形孢菌素敏感形式的蛋白激酶参与抑制Na+，K+，Cl-共转运的P-2-嘌呤受体的激活剂。(C)1998年Elsevier Science B.V.
This study examines the involvement of hormones and neuromediators in the regulation of Na+, K+, Cl- cotransport in renal epithelial cells using Madin-Darby canine kidney cells with low transepithelial electrical resistance (194 +/- 47 Omega/cm(2)). In this cell line, Na+, K+, Cl- cotransport measured as bumetanide-sensitive Rb-86 influx was inhibited up to 50-60% with agonists of P-2-purinoceptors (ATP approximate to ADP > UTP > AMY), slightly (15-30%) increased by activators of cAMP signaling (forskolin, 8-Br-cAMP) and was insensitive to activators of cGMP signaling (8-Br-cGMP, nitroprusside), EGF, angiotensin II, bradykinin, methacholine, propranolol, vasopressin, adenosine, dopamine and histamine. Thirty min of preincubation of MDCK cells with 0.1 mu M PMA completely blocked the activity of Na+, K+, Cl- cotransport whereas down-regulation of this enzyme by 24h of preincubation with 1 mu M PMA activated Na+, K+, Cl- cotransport by 60% and abolished the effect of short-term treatment with PMA. Regulation of Na+, K+, Cl- cotransport by ATP was insensitive to down-regulation of PMA-sensitive isoforms of protein kinase C. In addition, in inhibitor of protein kinase activity, staurosporine, abolished the effect of 0.1 mu M PMA but did not change inhibition of this carrier by ATP. Thus, these results show for the first time that P-2-purinoceptors and PMA-sensitive isoforms of protein kinase C play a key role in the regulation of Na+, K+, Cl- cotransport in MDCK cells. These results also show that neither PMA- nor staurosporine-sensitive forms of protein kinase are involved in the inhibition of Na+, K+, Cl- cotransport by activators of P-2-purinoceptors. (C) 1998 Elsevier Science B.V.